Intra-mammary teat sealant formulation and method of using same to reduce or eliminate visual defects in aged cheeses

ABSTRACT

Described is an intra-mammary teat sealant and a corresponding method of forming a physical barrier in the teat canal of a non-human animal for prophylactic treatment of mammary disorders during the animal&#39;s dry period. The method includes the step of infusing a bismuth-free teat seal formulation into the teat canal of the animal. The method also prevents the formation of black spot defect in dairy products, especially cheddar cheese, made from the milk of animals so treated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 11/869,966, filed Oct.10, 2007 now U.S. Pat. No. 7,906,138, which claims priority toprovisional application Ser. No. 60/850,572, filed Oct. 10, 2006,incorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to a bismuth-free, metal-containing,intra-mammary teat sealant to prevent mastitis in dry cows. Theintra-mammary teat sealant does not cause visual defects in dairy foods(especially cheese) made from milk from treated animals. The inventionis further directed to a method to prevent “black spot defect” (BSD) incheese.

BACKGROUND

Mastitis in dairy herds is one of the most costly and difficult diseasesencountered by dairy producers. Conventional therapies aimed at curingclinical mastitis include intra-mammary antimicrobial therapy. Despitethe commercial availability of numerous intra-mammary antimicrobialproducts, cure rates for clinical mastitis remain perplexingly low: 46%for Streptococcus spp., 21% for Staphylococcus spp., and only 9% forStaphylococcus aureus mastitis. See Wilson et al. (1996) NationalMastitis Council Proceedings 164-165, and Crandall et al. (2005) NMCAnnual Meeting Proceedings 215-216. Thus, dairy producers often managethe disease simply by culling mastitis-prone animals from their herds.

Because of the difficulty in treating mastitis, prevention of newintra-mammary infections is a major focus in the dairy industry. Therate of new infections is significantly higher during the dry period ascompared to new infections during the lactating period. (For example,one study showed that 61% of all new gram-negative intra-mammaryinfections occurred during the dry period. See Todhunter et al. (1995)J. Dairy Sci. 78:2366.) The three-week period immediately following dryoff; and the two weeks prior to calving, are periods particularly proneto new infections. Thus, in recent years dairy producers have focused aconsiderable amount of effort in “preventive maintenance” of cows duringtheir dry period.

In April of 2003, an internal (or “intra-mammary”) teat sealant (ITS)for use in dry cows was introduced in the US market. Marketed in the USunder the “ORBESEAL” trademark (US Trademark Registration Nos. 2,772,198and 3,120,693), the product was developed in New Zealand. The“ORBESEAL”-brand ITS introduced into the US market contains 65% w/wbismuth sub-nitrate dispersed in a viscous paste. The ITS product doesnot contain any antibiotics, nor does the product contain any activeantimicrobial agents. The ITS is injected into the teat end using atubular applicator syringe, in the same fashion as applying a dry cowantibiotic. The ITS product fills the fissures and folds of the teatcanal, thereby creating a physical barrier to pathogens. See U.S. Pat.No. 6,254,881, issued Jul. 3, 2001, incorporated herein by reference.

Initial studies of the “ORBESEAL”-brand product in New Zealand concludedthat the product functioned as well as a broad spectrum, long-actingintra-mammary antibiotic in preventing new intra-mammary infections atcalving and in preventing clinical appearance of mastitis through thefirst five (5) months of lactation. See Woolford et al. (1998) NewZealand Veterinary Journal 46:1. A more recent study in the US alsoconcluded that this ITS product improved the udder health of cowsalready infused with cloxacillin benzathine. See Godden et al. (2003) J.Dairy Sci. 86:3899-3911. Thus, the “ORBESEAL”-brand ITS has proven to bean effective tool in reducing the number of new cases of mastitis indairy cows during their dry period. Despite its relatively recentintroduction into the US market, the “ORBESEAL”-brand product hasenjoyed widespread market acceptance and is used extensively in US dairyherds. In short, the “ORBESEAL”-branded product is very good for itsintended purpose of preventing mastitis.

Subsequent to the introduction of the “ORBESEAL”-brand ITS product inthe US, a visual defect in aged dairy products, most notably agedcheddar cheeses, began to appear. The visual defect takes the form ofsmall, black spots (roughly 0.5 to 5 mm in diameter) that appearthroughout the aged cheese. The spots are a purely aesthetic, visualdefect that lowers the graded quality (and hence the market value) ofthe cheese affected with the problem. The spots are not accompanied byany organoleptic defect in the cheese. Cheese affected with the blackspots is saleable, albeit at a lower grade than unaffected cheeses. Thedefect has been termed “black spot defect” (BSD).

SUMMARY OF THE INVENTION

A first version of the invention is directed to a method of forming aphysical barrier in the teat canal of a non-human animal forprophylactic treatment of mammary disorders during the animal's dryperiod and simultaneously preventing BSD in dairy products made with theanimal's milk. The method comprises infusing teat seal formulation intothe teat canal of the animal, wherein the teat seal formulation isbismuth-free. The formulation is administered in an amount sufficient toform a physical barrier to entry of microorganisms into the teat, butdoes not cause black spot defect in diary products made with milk fromthe animal. The preferred embodiment of the method comprises infusing ateat seal formulation comprising a bismuth-free, non-toxic heavy metalsalt in a gel base. Preferably, the teat sealant is devoid ofanti-infective agents (i.e., the teat sealant preferably does notcontain antibiotics or other anti-infective active agents). Preferablythe method comprises infusing a teat seal formulation comprising atleast about 30% by weight of the bismuth-free, non-toxic heavy metalsalt, more preferably about 50% to about 75% by weight of thebismuth-free, non-toxic heavy metal salt, and more preferably stillabout 65% by weight of the bismuth-free, non-toxic heavy metal salt. Thepurpose of the salt is primarily to impart sufficient density to thecomposition so that the ITS “settles” into the teat canal. In oneversion of the method, the non-toxic heavy metal salt is selected fromthe group consisting of titanium dioxide, zinc oxide, barium sulfate andcombinations of these salts.

The gel base can be any suitable gel formulation, a host of which areknown in the pharmaceutical arts. The preferred gel base comprisesaluminum stearate and liquid paraffin (e.g. mineral oil, whitepetrolatum, yellow petrolatum, etc.). Typical gel bases include a wax oroil of some type, and a salt such as aluminum or magnesium stearate.

Another version of the invention is directed to an intra-mammary teatsealant comprising, in combination, a gel base, and a non-toxic heavymetal salt dispersed in the gel base, wherein the heavy metal salt isdevoid of bismuth. As noted earlier, the teat sealant preferablycomprises at least about 30% by weight, more preferably about 50% toabout 75% by weight, and more preferably still about 65% by weight ofthe heavy metal salt. The heavy metal salt preferably is titaniumdioxide, zinc oxide, barium sulfate or a combination of the same. Thegel base preferably comprises aluminum stearate and liquid paraffin.

Yet another version of the invention is an improvement to intra-mammaryteat sealants. Specifically, in a method of forming a physical barrierin the teat canal of a non-human animal for prophylactic treatment ofmammary disorders during the animal's dry period, wherein the methodcomprises the step of infusing a seal formulation into the teat canal ofthe animal without an anti-infective agent, the improvement of thepresent invention comprises infusing a teat seal formulation comprisinga bismuth-free, non-toxic heavy metal salt(s) in a gel base. Theimprovement prevents the formation of black spot defect in dairyproducts made from the milk of treated animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs of a typical black spot defect in an 18kg block of aged white cheddar cheese. FIG. 1A shows the surface of theblock, with a black spot defect readily visible. FIG. 1B is a magnifiedview (with a superimposed ruler) showing the dimensions of the defect.Such spots are equally distributed throughout the cheese.

FIG. 2 is an electron photomicrograph of a black spot defect exhibitingcharacteristic hair- or rod-like structures of bismuth (III) sulfidenanorods. No such structures appeared in any non-BSD cheeses or non-BSDcheese regions tested. The rods shown in FIG. 2 have diameters rangingfrom 37.09 nm to 129.33 nm.

FIG. 3 is an electron photomicrograph of a single rod-like structurefrom a black spot defect region. The rod shown in 130.88 nm in diameterand exhibits a line bisecting the length of the rod.

FIGS. 4A and 4B are images of laboratory-induced black spot defects. InFIG. 4A, the various components of “ORBESEAL”-brand intra-mammary teatsealant (ITS) were blended with cheese and each spot was photographedimmediately. FIG. 4B shows the same spots photographed after exposureeither to volatiles from aged cheddar cheese or hydrogen sulfide gas.Sites 4 and 5 contain bismuth subnitrate and the intact “ORBESEAL”-brandITS formulation, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Starting in late 2003, a number of inquiries have been made by cheesemakers to the University of Wisconsin-Madison, Department of FoodScience and Center for Dairy Research (CDR), seeking information aboutthe appearance of a novel “black spot defect” in aged cheeses, notablyaged cheddar cheese. Historically, grey to black discolorations incheese have been the result of several different and distinct causes,including the growth of specific microorganisms (e.g., certainenvironmental propionibacteria or molds) or the contamination of cheesewith food-grade lubricant debris. The particular BSD noted by the cheesemakers, however, did not fit the profile of a bacterial contaminant orother spoilage organism, nor did it appear to be lubricant debris thathad found its way into the milk stream or other debris introduced duringthe cheese-making process.

Thus, the first step was to determine the chemical structure of theblack spot defect. A great deal of effort was initially made to extractthe affected regions of BSD cheese. Extraction efforts using a widespectrum of organic solvents of varying polarity, hydrophobicity, etc.,proved fruitless as a means of isolating any type of organic pigmentmass from the cheese matrix. Although extraction of the BSD with organicsolvents was not successful, the extraction efforts did yield usefuldata. Notably, because the pigment did not dissolve or diffuse into suchsolvents, it could be concluded (with a high degree of probability) thatthe black spot pigment likewise would not dissolve or diffuse within thecheese matrix itself.

Visual examination of a growing number of cheese samples exhibiting thedefect (samples accumulated from commercial cheese makers) confirmedthis conclusion—the black spot pigment is well contained and does notappear to diffuse into the cheese matrix. See FIGS. 1A and 1B, which arephotographs of a typical 18 kg block of aged, white cheddar cheeseaffected with the BSD. FIG. 1A is a photograph of the outer surface ofthe cheese block. FIG. 1B is a magnified view of a single black spot,with a ruler superimposed on the image to show the dimensions of thespot. Spots such as the one shown in FIGS. 1A and 1B typically areequally distributed throughout the cheese block, ranging in size from <1mm to about 5 mm in diameter. The frequency of the spots within anygiven 18 kg cheese block varies widely, from <10 per block to well over100.

There were some anecdotal reports received from cheese makers thatspecific aging and storage strategies might aid in dissolving ordiffusing the spots to the point that they are no longer visuallynoticeable. (Because the defect is not accompanied by any organolepticdeficiency, “fading” the spots would ameliorate the condition.) Such aneffect, however, is highly unlikely given the stability of the pigmentto the organic solvents employed in the extraction efforts. In short,the extraction experiments performed by the present inventor usedsolvents having hydrophobicities similar to milk fat. If the black spotpigment dissolved or diffused into the cheese matrix itself (via anaging or storage protocol), the pigment should likewise readily dissolveor diffuse into an organic solvent having physical characteristicssimilar to milk fat. That result did not occur in the lab. Moreover,given the typical pH/acidic environment in cheese, and the typicalaging/shelf life periods associated with most aged cheddar-type cheeses(0.5 to 2 years), the anecdotal evidence that the defect can beameliorated via aging or storage protocols is without merit.

One experiment, however, proved most enlightening: the black spotpigment is readily dissolved in nitric acid. This strongly suggestedthat the pigment was an inorganic salt. Coupled with the timing of thefirst appearance of the defect, a working hypothesis was formulated,namely that the ITS was either a causative agent of (or at leastcorrelated with) the BSD. The discovery that the black spot pigmentreadily dissolves in acid supported a further hypothesis that thepigment may be comprised of bismuth III sulfide. Thus, it was concludedthe “ORBESEAL”-brand product, which in the US contains 65% by weight ofa bismuth-containing salt, was likely being inadvertently introducedinto the milk stream. As noted above, the “ORBESEAL”-brand product hasbeen commercially successful because it forms a tight physically barrierto the entry of pathogens into the teat canal. However, removing theproduct from a treated animal requires stripping of the animal's teats.It appeared that some of the ITS remained in the teats after strippingand was finding its way into the cheese milk.

The next phase of research operated pursuant to a hypothesis thatbismuth III sulfide was in fact the causative agent of the BSD. Bismuthsubnitrate itself is white and relatively chemically inert. Thus, itstrace presence in fluid milk, mozzarella cheese, and yogurt is notreadily apparent visually. However, in aged cheeses with high flavorintensity, the black spot defect appears prominently. Thus, it washypothesized that bismuth III sulfide (a black, relatively insolublesalt) was the product of a reaction between bismuth subnitrate (from theITS) and hydrogen sulfide produced within the aging cheese by theactions of ripening microflora, enzymes, and certain cofactors acting onthe protein/amino acid components of cheese.

In short, the hypothesis was that bismuth subnitrate made its way intothe milk stream due to incomplete removal of the ITS prior to milking.The bismuth subnitrate then reacted with hydrogen sulfide to yieldbismuth III sulfide according to Equation 14BiNO₃(OH)₂BiO(OH)+H₂S→Bi₂S₃(insol., black)  (Eq. 1)The product, bismuth Ill sulfide (or simply bismuth sulfide) is arelatively insoluble, black salt.

In addition to having a specific elemental target, bismuth, it washypothesized that, under the conditions or chemical environment presentwithin the cheese matrix, the Bi₂S₃ molecules would form a crystallinestructure referred to in the literature as nanorods or nanowhiskers. SeeW. Zhang et al. (2001) Sol. State Comm. 119:143-146 and B. Zhang et al.(2006) J. Phys. Chem. 110:8978-8985. These bismuth-containing nanorodswould thus constitute light-diffracting particles capable of impartingthe grey to black hue seen in the black spot defect.

Efforts were then focused on confirming: 1) the elemental presence ofbismuth in the black spot defects; and 2) confirming the physicalpresence of bismuth Ill sulfide nanorod structures within the black spotdefects.

Confirming the presence of bismuth within the black spots wasinvestigated using inductively coupled plasma mass spectroscopy (ICPMS).AOAC International (Association of Analytical Communities) method 993.14was used. The first efforts screened multiple black spots for thepresence of several elements that could be contributing to BSD. Theinitial experiments focused on metal salts/oxides typical of those foundin milk- and cheese-handling/conveying equipment, and other residualmetal derivatives present in food-grade processing. As a measure ofcontrol, cheese compositional analyses were conducted. Specifically,protein, ash, and moisture were measured using methods 2001.14, 935.42,and 926.08, of the Official Methods of Analysis, AOAC 17^(th) Edition,respectively (copyright 2000, ISBN: 0935584-67-6). Fat was measuredaccording to the method described in the Official Methods of Analysis,AOAC 17th Edition.

Transmission electron microscopy (TEM) studies were performed asfollows: approximately 100 μl double-distilled water was added tosamples and the mixture was pulverized into a suspension with a glassrod. Approximately 5 μl aliquots of suspended sample were deposited ontopolyvinyl alcohol-formaldehyde acetal-coated 300 mesh copper TEM grids(Ted Pella, Inc., Redding, Calif.). Excess sample was wicked away withsmall sections of filter paper and the remaining sample was dried to thesurface of the grid at room temperature. In some cases, NANO-W-brand TEMnegative stain (Nanoprobes, Incorporated, Yaphank, N.Y.) was appliedover the dried sample to enhance contrast and visibility. Specimens wereobserved with a Philips CM 120 electron microscope and images werecollected with a MegaView 3 Digital camera (from SIS, Ringoes, N.J.).Measurements were taken with SIS-brand analysis software (Ringoes, N.J.)calibrated with reference samples of known lengths.

ICPMS results demonstrated the presence of the elements chromium,copper, iron, nickel, and bismuth in the BSD region. Althoughincremental increases in the elements chromium, copper, iron, and nickelwere found, bismuth concentrations in the BSD region were routinelythree orders of magnitude greater than the same cheese assayed innon-BSD areas. These results show that bismuth is the only elementpresent in sufficient quantities to participate in a pigment-generatingreaction.

Several hundred TEM images of BSD regions of cheese samples werecaptured with a single, consistent conclusion. Nanorods typical of thosereported in the literature cited above were uniquely present in the BSDcheese region. An example of such an image is presented in FIG. 2. Thenanorod structures shown in FIG. 2 are too small to be readily detectedwith a light microscope. The nanorods shown in FIG. 2 range in diameterfrom about 69 nm to about 130 nm. The nanorods are very stable to thepotentially abusive conditions of TEM. The rods appear to have aslightly mottled surface and they exhibit a characteristic line runningthe length of the nanorod. See FIG. 3, which is an increasedmagnification view of a single nanorod. The presence of such structuresis consistent with the presence of bismuth sulfide nanorods formed underthe conditions present in the cheese matrix.

To confirm the reactivity of bismuth subnitrate as a reactant in formingbismuth Ill sulfide nanorods, additional assays were conducted to see ifBSD could be purposefully recreated in the lab. In short, cheeses weremanufactured with known amounts of ITS components and subjected toeither the authentic volatile gasses produced by maturing cheeses orexposed directly to the hypothesized bismuth subnitrate co-reactant,hydrogen sulfide gas. In both situations, the responses were invariablythe same: When cheese samples containing bismuth subnitrate or thecomplete ITS formulation were exposed to authentic cheese volatiles orto “chemical standard”-grade H₂S gas, each formed identical blackpigmentation with the accompanying presence of nanorod structures,further confirming that bismuth subnitrate is the culprit in BSD. Theresults are shown in FIG. 4. No other ITS component formed black spotswhen so exposed. Furthermore, the susceptible sites formed identicallyblack pigmentation when exposed to either authentic cheese volatiles orto hydrogen sulfide gas, thus confirming that hydrogen sulfide gas wasthe suspected co-reactant.

From a cheese manufacturing and aging or ripening standpoint, it is notreasonable to consider targeting the elimination of hydrogen sulfide gasproduction as a means of controlling BSD. Hydrogen sulfide is a highlyaroma active compound, the product of microbial, enzymatic and co-factoractivities against sulfur-containing amino acids such as cysteine. SeeArfi et al., (2002) Appl. Microbiol. Biotechnol. 58:503-510. There isample research to support the claim that hydrogen sulfide gas is anecessary and/or valued component of typical aged cheddar cheese flavor.See Burbank & Qian (2005) J. Chrom. 1066:149-157. Even if a scheme wasdevised to eliminate the production of hydrogen sulfide (by interruptingof dozens of complex metabolic pathways) the resulting final productruns the risk of a flavor character unacceptable to cheese graders andconsumers.

Thus, in the present invention, the formulation of the ITS is altered toexclude bismuth-containing salts. As shown in the above experiments, itis the bismuth subnitrate in the commercially-available ITS that giverise to the BSD. Thus, using an ITS that does not contain bismuth, norany other heavy metal salt that reacts with hydrogen sulfide to yield adark, insoluble pigment, eliminates the BSD.

EXAMPLES

The following Examples are included solely to provide a more completedescription of the invention disclosed and claimed herein. The Examplesdo not limit the invention in any way.

Example 1

A test ITS according to the present invention was formulated. The testITS was identical to the “ORBESEAL”-brand formulation, with theexception that it did not contain any bismuth or bismuth-containingsalts. The test ITS comprised zinc oxide, titanium dioxide, mineral oil(30-40%), and aluminium stearate.

To prepare a batch of ITS, liquid paraffin (e.g., mineral oil) isdelivered into a suitable vessel equipped with a mixer. Aluminumstearate is added and the mixture is stirred and heated to about 160° C.until homogeneous (about two hours). The non-toxic, non-bismuthcontaining salt is then added in portions to the mixture, with stirring,until the desired amount of metal salt has been added. The mixture isthen stirred until homogenous. The products is then transferred intoconventional injector tubes for intra-teat administration.

Example 2

The object of this Example was to compare retention within the teats ofnon-lactating dairy cows of an ITS according to the present invention ascompared to the “ORBESEAL”-brand product.

The study was performed at the Blaine Dairy of the University ofWisconsin-Madison (UW), in Arlington, Wis. Sixteen (16) cows (n=64teats) were enrolled on the day of dry off. All enrolled cows wererequired to have four functional quarters and no visible sign ofmastitis. All cows were dried off and received intramammary antibioticdry cow therapy (DCT) according to standard UW dairy herd protocols.Parity and milk yield (at dry off) were recorded for each cow. Uponinitial enrolment, teats were scored for shape, length, diameter anddegree of teat end hyperkeratosis. Within each cow, two teats wereassigned to receive the “ORBESEAL”-brand ITS and two teats were assignedto receive the test ITS. The administration protocol was designed toensure that each product was administered uniformly among teatlocations, eight teats each per product administered in each location(right-rear, right-front, left-rear, left-front). Sealant tubes wereweighed before and after administration to determine the net volumeadministered. Prior to receiving DCT & the internal teat sealant, teatends were cleaned using a single 70% isopropanol alcohol wipe andpartial insertion technique was used to reduce the probability ofintroducing teat skin pathogens. After administration of the internalsealant, teats were dipped with an external teat disinfectant.

Teats were examined on Days 1, 2, 3, 4, 5, 6, 7, 14, 28, 42 and atcalving to detect redness, swelling and/or sealant leakage. On Days 14,28, 42 and at calving, sealant was removed from one teat (eight teatsfor each sealant per removal day) of each cow by hand stripping. Theremoved sealant was collected with the first milk into graduated 50 mlplastic vials. The vials were centrifuged (3000 rpm×5-7 minutes), thesupernatant rinsed, and the recovered sealant weighed. The amount ofrecovered sealant was compared at each period between the testITS-treated teats and the “ORBESEAL”-brand ITS-treated teats. Follow upsamples were collected at Day 1 post-calving using the same procedure.At all sampling periods, after removal of the sealant, teats were dippedwith an external teat disinfectant. After calving, quarter milk sampleswere aseptically collected from all quarters and cultured to identifyintramammary infections.

Group Characteristics—Teat Length and Volume:

A total of 16 cows were enrolled into the study for a total number of 64teats; 32 teats received the test ITS and 32 teats received the“ORBESEAL”-brand ITS. The teat length and volume for the test populationis shown in Table 1:

TABLE 1 Mean, Standard Deviation, and Standard Errors for Teat Lengthand Volume by Compound Group A (“ORBESEAL”-ITS) Group B (Test ITS) NMean S.D. S.E. N Mean S.D. S.E. P Length 32 5.12 0.88 0.16 32 5.11 0.860.15 0.95 Volume 32 23.60 8.61 1.52 32 25.72 9.73 1.73 0.36

There was no significant difference in teat length or volume for teatsin Group A or Group B (p>0.36). Overall teat length was 5.11 cm, rangingfrom 3.3 cm to 7.3 cm. The average teat length was 5.12 cm for Group Aand 5.11 cm for Group B, and ranged from 3.3 cm to 7.3 cm for Group Aand from 3.5 cm to 7.10 cm for Group B. A two Sample paired t-test wasperformed to test the null hypothesis that the mean teat length in thetwo treatment group did not differ. There was no significant differencein teat length between teats randomized to receive either product(p=0.95).

The overall teat volume was 24.66 cm³, ranging from 12.47 cm³ to 54.34cm³ (std. dev. 9.17 cm³). The mean teat volume was 23.6 cm³, rangingfrom 12.54 cm³ to 54.34 cm³ (std. dev. 8.60 cm³) in Group A. In Group B,mean teat volume was 25.71 cm³, ranging from 12.47 cm³ to 48.25 cm³(std. dev. 9.73 cm³). A two sample paired t-test was used to test thenull hypothesis that the teat volume in the two groups did not differ.There was no significant difference in teat volume between teatsrandomized to receive either product (p=0.95 and p=0.35; log transformedanalysis).

Hyperkeratosis: Teat-end health was scored for hyperkeratosis using thefollowing scale: No ring (N), Smooth Ring (S), Rough (R), Very Rough(VR). The distribution of teat scores was: N (n=21; 32.8%), S (n=31;48.4%), R (n=11; 17.2%) and VR (n=1, 2%). An X² test confirmed that thedistribution of hyperkeratosis score was not associated with treatmentgroup (p=0.13).

TABLE 2 Descriptive Statistics for Hyperkeratosis Group A Group B(“ORBESEAL” ITS) (Test ITS) Overall Score Frequency Percent FrequencyPercent Frequency Percent N 13 40.63 8 25.00 21 32.81 S 11 34.38 2062.50 31 48.88 R 7 12.88 4 12.50 11 17.19 VR 1 3.23 n/a n/a 1 1.56

Amount of Sealant Administered, Recovered and Lost: Statistical analysesusing a paired t-test were performed to determine if the amount ofsealant administered, recovered, or lost (not recovered) did not differbased on treatment group.

TABLE 3 Two Sample Paired t-Test for the Mean of Administered, Recoveredand Lost Sealant by Product Group A Group B (“ORBESEAL” ITS) (Test ITS)Overall N Mean S.D. N Mean S.D. N Mean S.D. P Administered 32 3.46 0.8532 3.77 0.96 64 3.62 0.91 0.12 Recovered 32 0.85 1.42 32 0.79 1.57 640.82 1.49 0.89 Lost 32 2.71 1.37 32 3.04 1.55 64 2.88 1.46 0.40

Of the four (4) grams in each tube, the overall amount of sealantadministered was 3.62 grams. There was no significant difference in theamount of “ORBESEAL”-brand ITS (3.46 gram) or test ITS (3.62)administered (P=0.12).

Overall, the amount of sealant recovered was 0.82 gram and there were nosignificant differences based on treatment (P=0.89). The overall amountof sealant lost was 2.88 grams and did not differ by treatment group(P=0.40). The amount of sealant recovered tended to be associated withrecovery date (P=0.08) with more sealant recovered on day 14 as comparedto other recovery periods (day 14, recovery=1.6 grams; day 28recovery=0.68 grams; day 42 recovery=0.65 grams; calving recovery=0.33grams).

Simple linear regression was used to determine that there was nosignificant relationship between the amount of administered sealant andteat volume (p=0.59, p=0.53).

For the recovered sealant a simple linear regression test was performedto test the null hypothesis that there was no significant linearrelationship between the amount of recovered sealant and the teatvolume. Only 6% of the recovered sealant was accounted for by teatvolume (P=0.05).

The proportion of administered sealant was not significantly associatedwith teat volume, while the recovered sealant was correlatedsignificantly with the teat volume but only for a small proportion (6%).

A one-way ANOVA was used to determine univariate relationships betweenthe amount of administered and recovered sealant and the teat position,the product, the cow.

TABLE 4 Administered and Recovered Volume Univariate Association Table(P values) Administered Recovered (p) (p) Teat volume 0.53 0.13 Teatposition 0.56 0.52 Product 0.19 0.88 Day — 0.07 Cow 0.05 0.17

What is claimed is:
 1. A method of forming a physical barrier in theteat canal of a non-human animal for prophylactic treatment of mammarydisorders during the animal's dry period wherein the physical barrierdoes not cause black spot defect in dairy products made with milk fromthe animal, the method comprising: infusing an amount of a teat sealformulation into the teat canal of the animal, wherein the teat sealformulation comprises at least about 30% by weight of a bismuth-free,non-toxic, inorganic heavy metal salt in a gel base, and wherein theamount is sufficient to form a physical barrier to entry ofmicroorganisms into the teat canal, and wherein the teat sealformulation does not cause black spot defect in aged cheese made withmilk from the animal.
 2. The method of claim 1, wherein the teat sealformulation is devoid of anti-infective agents.
 3. The method of claim1, wherein the teat seal formulation comprises about 50% to about 75% byweight of the salt.
 4. The method of claim 1, wherein the teat sealformulation comprises about 65% by weight of the salt.
 5. The method ofclaim 1, wherein the gel base comprises aluminum stearate.
 6. The methodof claim 1, wherein the gel base comprises liquid paraffin.